Drying and cicatrizing ocular surface diseases can be debilitating and, in advanced disease states, cause blindness. Goblet cells, mucin-secreting cells that are intercalated within the stratified epithelium of the conjunctiva disappear in these diseases, as the epithelium becomes keratinized. Little is known regarding factors regulating goblet cell differentiation in the conjunctiva. With this application, we present evidence that mice null for SAM pointed domain Ets transcription factor, Spdef, lack conjunctival goblet cells. We further demonstrate by microarray comparison of fornicial conjunctiva of Spedf-null and wild type mice that, in addition to downregulation of goblet cell-specific genes, there is a significant downregulation of 4 genes in the Wnt signaling pathway. Of these, Sfrp3 (Frzb), an antagonist of canonical Wnt signaling, is downregulated 115 fold. Immunofluorescence microscopy indicates that the antagonist is expressed in the conjunctival goblet cell, raising the possibility that the goblet cell secretes the protein to effect epithelial homeostasis and/or goblet cell differentiation. Based on these findings, we propose the following specific aims to test hypotheses relevant to goblet cell function and differentiation at the ocular surface. Aim 1. Determine the effect of lack of goblet cells on ocular surface health in the Spdef-null mice as indicated by comparison of tear volume, fluorescein staining, exposure to dry eye chamber conditions and susceptibility to infection to that of wild type mice. Aim 2. Compare expression levels of genes down/upregulated in fornicial conjunctiva of Spdef-null mice (goblet cell mucin biosynthesis genes, Wnt signaling pathway genes and epithelial differentiation genes) to RNA from fornicial conjunctiva of mice with smoke induced goblet cell differentiation to verify genes in goblet cell differentiation/function as opposed to those involved in epithelial response to lack of goblet cells. Aim 3. Examine the role of Wnt signaling in goblet cell/conjunctival cell differentiation by determining if the goblet cell is the source of the Wnt inhibitor Sfrp3 identified by microarray analysis as downregulated in Spdef null mice and by assaying the function of the soluble Wnt inhibitor in Sfrp3 (Frzb) null mice and in vitro. Aim 4. Determine whether goblet cell differentiation can be induced by overexpression of SPDEF in conjunctival epithelial cell culture and whether downregulation of the Wnt pathway influences goblet cell differentiation in vitro. Aim 5. Determine whether expression of Spdef, the Wnt pathway antagonist Sfrp3 identified as downregulated in the absence of Spdef, and the epithelial differentiation gene Sprr2h that is upregulated in Spdef-/- mice, are altered in human dry eye diseases where goblet cell numbers are reduced. PUBLIC HEALTH RELEVANCE: Drying eye diseases often cause irritation and poor vision but in severe forms cause blindness. Research in this application seeks to learn the cellular and molecular changes that occur in the outer ocular surface cell layers that are associated with the diseases. With these insights future therapies may be developed.